Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive

In mammals, a subset of genes inherit gametic marks that establish parent of origin-dependent expression patterns in the soma ([1] and references therein). The currently most extensively studied examples of this phenomenon, termed genomic imprinting, are the physically linked Igf2 (insulin-like growth factor II) and H19 genes, which are expressed mono-allelically from opposite parental alleles [1] [2]. The repressed status of the maternal Igf2 allele is due to cis elements that prevent the H19 enhancers [3] from accessing the Igf2 promoters on the maternal chromosome [4] [5]. A differentially methylated domain (DMD) in the 5' flank of H19 is maintained paternally methylated and maternally unmethylated [6] [7]. We show here by gel-shift and chromatin immunopurification analyses that binding of the highly conserved multivalent factor CTCF ([8] [9] and references therein) to the H19 DMD is methylation-sensitive and parent of origin-dependent. Selectively mutating CTCF-contacting nucleotides, which were identified by methylation interference within the extended binding sites initially revealed by nuclease footprinting, abrogated the H19 DMD enhancer-blocking property. These observations suggest that molecular mechanisms of genomic imprinting may use an unusual ability of CTCF to interact with a diverse spectrum of variant target sites, some of which include CpGs that are responsible for methylation-sensitive CTCF binding in vitro and in vivo.     

Nucleosome positioning signals in the DNA sequence of the human and mouse H19 imprinting control regions

We have investigated the sequences of the mouse and human H19 imprinting control regions (ICRs) to see whether they contain nucleosome positioning information pertinent to their function as a methylation-regulated chromatin boundary. Positioning signals were identified by an in vitro approach that employs reconstituted chromatin to comprehensively describe the contribution of the DNA to the most basic, underlying level of chromatin structure. Signals in the DNA sequence of both ICRs directed nucleosomes to flank and encompass the short conserved sequences that constitute the binding sites for the zinc finger protein CTCF, an essential mediator of insulator activity. The repeat structure of the human ICR presented a conserved array of strong positioning signals that would preferentially flank these CTCF binding sites with positioned nucleosomes, a chromatin structure that would tend to maintain their accessibility. Conversely, all four CTCF binding sites in the mouse sequence were located close to the centre of positioning signals that were stronger than those in their flanks; these binding sites might therefore be expected to be more readily incorporated into positioned nucleosomes. We found that CpG methylation did not effect widespread repositioning of nucleosomes on either ICR, indicating that allelic methylation patterns were unlikely to establish allele-specific chromatin structures for H19 by operating directly upon the underlying DNA-histone interactions; instead, epigenetic modulation of ICR chromatin structure is likely to be mediated principally at higher levels of control. DNA methylation did, however, both promote and inhibit nucleosome positioning at several sites in both ICRs and substantially negated one of the strongest nucleosome positioning signals in the human sequence, observations that underline the fact that this epigenetic modification can, nevertheless, directly and decisively modulate core histone-DNA interactions within the nucleosome.     

YY1's role in DNA methylation of Peg3 and Xist

Unusual clusters of YY1 binding sites are located within several differentially methylated regions (DMRs), including Xist, Nespas and Peg3, which all become methylated during oogenesis. In this study, we performed conditional YY1 knockdown (KD) to investigate YY1''s roles in DNA methylation of these DMRs. Reduced levels of YY1 during spermatogenesis did not cause any major change in these DMRs although the same YY1 KD caused hypermethylation in these DMRs among a subset of aged mice. However, YY1 KD during oogenesis resulted in the loss of DNA methylation on Peg3 and Xist, but there were no changes on Nespas and H19. Continued YY1 KD from oogenesis to the blastocyst stage caused further loss in DNA methylation on Peg3. Consequently, high incidents of lethality were observed among embryos that had experienced the reduced levels of YY1 protein. Overall, the current study suggests that YY1 likely plays a role in the de novo DNA methylation of the DMRs of Peg3 and Xist during oogenesis and also in the maintenance of unmethylation status of these DMRs during spermatogenesis.     

Maternal-specific footprints at putative CTCF sites in the H19 imprinting control region give evidence for insulator function

Parent-of-origin-specific expression of the mouse insulin-like growth factor 2 (Igf2) gene and the closely linked H19 gene are regulated by an intervening 2 kb imprinting control region (ICR), which displays parentspecific differential DNA methylation [1] [2]. Four 21 bp repeats are embedded within the ICR and are conserved in the putative ICR of human and rat Igf2 and H19, suggesting that the repeats have a function [3] [4]. Here, we report that prominent DNA footprints were found in vivo on the unmethylated maternal ICR at all four 21 bp repeats, demonstrating the presence of protein binding. The methylated paternal ICR displayed no footprints. Significantly, the maternal-specific footprints were localized to putative binding sites for CTCF, a highly conserved zinc-finger DNA-binding protein with multiple roles in gene regulation including that of chromatin insulator function [5] [6]. These results strongly suggest that the maternal ICR functions as an insulator element in regulating mutually exclusive expression of Igf2 and H19 in cis.     

Boundaries between chromosomal domains of X inactivation and escape bind CTCF and lack CpG methylation during early development

Escape from X inactivation results in expression of genes embedded within inactive chromatin, suggesting the existence of boundary elements between domains. We report that the 5' end of Jarid1c, a mouse escape gene adjacent to an inactivated gene, binds CTCF, displays high levels of histone H3 acetylation, and functions as a CTCF-dependent chromatin insulator. CpG island methylation at Jarid1c was very low during development and virtually absent at the CTCF sites, signifying that CTCF may influence DNA methylation and chromatin modifications. CTCF binding sites were also present at the 5' end of two other escape genes, mouse Eif2s3x and human EIF2S3, each adjacent to an inactivated gene, but not at genes embedded within large escape domains. Thus, CTCF was specifically bound to transition regions, suggesting a role in maintaining both X inactivation and escape domains. Furthermore, the evolution of X chromosome domains appears to be associated with repositioning of chromatin boundary elements.     

Critical DNA binding interactions of the insulator protein CTCF: a small number of zinc fingers mediate strong binding, and a single finger-DNA interaction controls binding at imprinted loci

The DNA-binding protein CTCF (CCCTC binding factor) mediates enhancer blocking insulation at sites throughout the genome and plays an important role in regulating allele-specific expression at the Igf2/H19 locus and at other imprinted loci. Evidence is also accumulating that CTCF is involved in large scale organization of genomic chromatin. Although CTCF has 11 zinc fingers, we show here that only 4 of these are essential to strong binding and that they recognize a core 12-bp DNA sequence common to most CTCF sites. By deleting individual fingers and mutating individual sites, we determined the orientation of binding. Furthermore, we were able to identify the specific finger and its point of DNA interaction that are responsible for the loss of CTCF binding when CpG residues are methylated in the imprinted Igf2/H19 locus. This single interaction appears to be critical for allele-specific binding and insulation by CTCF.     

CTCF-binding sites within the H19 ICR differentially regulate local chromatin structures and cis-acting functions

It is generally assumed that CTCF-binding sites are synonymous with the demarcation of expression domains by promoting the formation of chromatin loops. We have proposed earlier, however, that such features may be context-dependent. In support of this notion, we show here that chromatin loop structures, impinging on CTCF-binding sites 1/2 and 3/4 at the 5′ and 3′-ends, respectively, within the maternal allele of the H19 imprinting control region (ICR), differ significantly. Although abrogation of CTCF binding to the maternal H19 ICR allele results in loss of chromatin loops in the 3′-region, there is a dramatic gain of long-range chromatin loops impinging on the 5′-region. As the degree of occupancy of its four CTCF-binding sites discriminates between the chromatin insulator and replication timing functions, we submit that the CTCF-binding sites within the H19 ICR are functionally diverse and organize context-dependent higher order chromatin conformations.     

Chromosome-wide analysis of parental allele-specific chromatin and DNA methylation

To reveal the extent of domain-wide epigenetic features at imprinted gene clusters, we performed a high-resolution allele-specific chromatin analysis of over 100 megabases along the maternally or paternally duplicated distal chromosome 7 (Chr7) and Chr15 in mouse embryo fibroblasts (MEFs). We found that reciprocal allele-specific features are limited to imprinted genes and their differentially methylated regions (DMRs), whereas broad local enrichment of H3K27me3 (BLOC) is a domain-wide feature at imprinted clusters. We uncovered novel allele-specific features of BLOCs. A maternally biased BLOC was found along the H19-Igf2 domain. A paternal allele-specific gap was found along Kcnq1ot1, interrupting a biallelic BLOC in the Kcnq1-Cdkn1c domain. We report novel allele-specific chromatin marks at the Peg13 and Slc38a4 DMRs, Cdkn1c upstream region, and Inpp5f_v2 DMR and paternal allele-specific CTCF binding at the Peg13 DMR. Additionally, we derived an imprinted gene predictor algorithm based on our allele-specific chromatin mapping data. The binary predictor H3K9ac and CTCF or H3K4me3 in one allele and H3K9me3 in the reciprocal allele, using a sliding-window approach, recognized with precision the parental allele specificity of known imprinted genes, H19, Igf2, Igf2as, Cdkn1c, Kcnq1ot1, and Inpp5f_v2 on Chr7 and Peg13 and Slc38a4 on Chr15. Chromatin features, therefore, can unequivocally identify genes with imprinted expression.